Blockade of DDR1/PYK2/ERK signaling suggesting SH2 superbinder as a novel autophagy inhibitor for pancreatic cancer

Pancreatic cancer is highly lethal, of which 90% is pancreatic ductal adenocarcinoma (PDAC), with a 5-year survival rate of less than 12%, lacking effective treatment options and late diagnosis. Furthermore, the tumors show an intense resistance to cytotoxic chemotherapies. As autophagy is elevated in PDAC, targeting the autophagic pathway is regarded as a promising strategy for cancer treatment. Immunofluorescence and transmission electron microscopy were utilized to assess the autophagic flux. Label-free quantitative phosphoproteomics was used to figure out critically altered tyrosine phosphorylation of the proteins. Tumor-bearing mice were used to validate that SH2 TrM-(Arg)9 restrained the growth of tumor cells. SH2 TrM-(Arg)9 inhibited collagen-induced autophagy via blocking the DDR1/PYK2/ERK signaling cascades. SH2 TrM-(Arg)9 improved the sensitivity of PANC-1/GEM cells to gemcitabine (GEM). Inhibition of autophagy by SH2 TrM-(Arg)9 may synergized with chemotherapy and robusted tumor suppression in pancreatic cancer xenografts. SH2 TrM-(Arg)9 could enter into PDAC cells and blockade autophagy through inhibiting DDR1/PYK2/ERK signaling and may be a new treatment strategy for targeted therapy of PDAC.


INTRODUCTION
Pancreatic cancer (PC) is a highly fatal malignancy in the digestive system, threatening hundreds of thousands of lives worldwide with a 5-year overall survival rate of only 12% [1].Approximately 90% of pancreatic malignancies are pancreatic ductal adenocarcinomas (PDAC) originated in the pancreatic ductal epithelium [1,2].Many patients with PC are already in the middle or advanced stages when diagnosed due to the unique location and function of the pancreas; therefore, surgical treatment alone is not effective.Chemotherapy is usually the mainstay in treating advanced PDAC.Gemcitabine (GEM), combined with paclitaxel or FOLFIRINOX (fluorouracil, irinotecan, leucovorin, and oxaliplatin), is currently the first-line chemotherapy strategy [3].However, the effectiveness of chemotherapy for advanced PDAC is limited due to chemoresistance and side effects [2].Other treatment options, such as targeted therapy and immunotherapy, slow down the development of PDAC to a certain extent, but there is no substantial advantage over chemotherapy [4,5].Additionally, some of these treatment options only benefit a small group of patients with specific genotype [3].Therefore, it is necessary to find new therapeutics with higher efficacy, wider beneficiaries, and less drug resistance.
Autophagy is a self-conserving process in which cells recycle biomolecules and damage organelles under stress or starvation conditions.Autophagy produces intermediate metabolites for biosynthesis and energy production, and promotes tumor growth by degrading and recycling damaged DNA, misfolded proteins, and intracellular pathogens [6].Abnormal activation of autophagy flux exists in PDAC tumor tissues, cancer cells, and stem cells [7,8].Autophagy of other cells in the PDAC tumor microenvironment (such as pancreatic stellate cells) also provides energy for tumor growth and development [9].Many studies have shown that the increased level of autophagy may be an essential cause of PDAC development [10,11].Therefore, inhibition of autophagy is thought to be an important strategy to treat PDAC.To date, small-molecule drugs that inhibit autophagy and novel chelators have shown sound anticancer effects in vitro and in vivo [12,13].
The SH2 domain is a conserved sequence that can specifically bind to phosphorylated tyrosine (pY), and regulate various cellular processes by affecting the interaction between proteins [14].Abnormal pY levels of proteins play an important role in the cancer development [15,16].The SH2 superbinder, a triple mutant of the SH2 domain, has a stronger binding ability for pY than the wild-type SH2 domain [17].A previous study has proved that SH2 superbinder can capture a variety of pY proteins, blockade multiple signaling pathways, and suppress the growth of PDAC cells [18].Therefore, the SH2 superbinder (termed SH2 TrM-(Arg)9 in the following contents) is expected to be a candidate drug for the treatment of PDAC.Meanwhile, we found that the SH2 superbinder could inhibit the autophagy of PDAC cells, but the precise mechanism remained undisclosed.
The collagen receptor DDR1 (discoidin domain receptor 1) is a member of the receptor tyrosine kinase (RTK) family which mediates the migration and proliferation of several cell types [19].DDR1 displays sustained activation upon interaction with collagen and the high expression level of DDR1 is significantly correlated with poor PDAC survival, implying that collagen/DDR1 mediated signaling would play an important role in cancer progression [20].However, the relationship between DDR1 and autophagy has been poorly reported.
In this study, we uncovered a mechanism driven by DDR1/PYK2/ ERK signaling that controls PDAC therapy resistance, expecting to provide new research ideas and treatment strategies for targeted therapy of PDAC.
As the DDR1/JAK/STAT signaling pathways are involved in cancer progression [21], we evaluated whether the vital proteins would be affected by SH2 TrM-(Arg)9.Up to now, DDR1 has gotten less attention compared to other RTKs, such as EGFR, VEGFR et al.Besides, in the existing researches, Y513 of DDR1 is critical for the DDR1 mediated signaling, which functions involved cell migration, invasion, stem cell differentiation and et al. [22][23][24][25].Y792 is another phosphorylation site located in kinase domain of DDR1, which may affect the kinase activity of DDR1 [26].But little is known about the function of this site.In this paper, we evaluated the effect of SH2 TrM-(Arg)9 on Y513 and Y792 of DDR1, and it was interesting that SH2 TrM-(Arg)9 only restrained phosphorylation of Y792, but not Y513 of DDR1 (Fig. 3).The pSTAT3 and pJAK2 were decreased after SH2 TrM-(Arg)9 treatment, whereas the DDR1(pY513) showed no obvious change (Fig. 1L).These data revealed SH2 TrM-(Arg)9 might lead to cell death progression by blocking the JAK/STAT signaling pathway in PDAC cells without affecting DDR1(Y513).
SH2 TrM-(Arg)9 suppressed autophagy in PANC-1 Cells Our previous data [17] have shown that SH2 TrM-(Arg)9 possesses anti-tumor activity and has a wide range of mechanisms.However, the relationship between SH2 TrM-(Arg)9 and autophagy is unclear.We used transmission electron microscopy to observe the autophagosomes and autolysosomes, and to investigate whether SH2 TrM-(Arg)9 would influence autophagy in PDAC cells.Consistent with previous study [27], PDAC cells showed elevated autophagy under basal conditions.As shown in Fig. 2A, B, the number of autophagosomes was markedly increased in PANC-1 cells treated with SH2 TrM-(Arg)9, but not in the control groups.Next, we analyzed the levels of LC3, p62, and Beclin1.The results showed SH2 TrM-(Arg)9 may induce the accumulation of LC3 (Fig. 2C).Meanwhile, levels of p62 increased, but Beclin1 showed no obvious changes (Fig. 2C).This observation also confirmed SH2 TrM-(Arg)9 markedly increased the number of GFP-LC3-positive puncta transiently transfected with GFP-LC3 constructs (Fig. 2D, E).
The increased levels of LC3-II or autophagic vacuoles are insufficient evidence to demonstrate autophagic flux induction, as they can be a result of an increase in their formation either from this induction or from the inhibition of autophagic degradation [28].In this regard, we demonstrated SH2 TrM-(Arg)9 had no significant effect on the expression levels of autophagy core genes.The increased LC3-II levels after SH2 TrM-(Arg)9 treatment were further increased by chloroquine (CQ).
CQ, a clinically available antimalarial agent, blocks late-stage autophagy by impairing lysosomal acidification and has been used to inhibit autophagy in patients [29,30].Compared with CQ, SH2 TrM-(Arg)9 increased LC3-II to a higher level and further enhanced LC3-II levels in cells pre-treated with CQ (Fig. 2F).These data indicated that increased LC3-II levels in SH2 TrM-(Arg)9treated cells were unlikely due to the enhanced autophagic flux but the suppression of the late maturation and degradation stages.We further used immunofluorescence staining of LAMP2 and LC3 to confirm this finding.The images displayed SH2 TrM-(Arg)9 impaired autolysosome formation (Fig. 2G, H).Meanwhile, p62 increased, and there was no change in Beclin1 after cells were stimulated with SH2 TrM-(Arg)9 (Fig. 2F).These findings suggested SH2 TrM-(Arg)9 blocked autophagic flux in a similar way to CQ and exhibited a stronger inhibitory effect on autophagy.
Inhibition of the mTOR-mediated signaling pathway plays an essential role in promoting autophagy [32].To investigate whether the mTOR-mediated signaling pathway would be affected during the SH2 TrM-(Arg)9-inhibited autophagy process, we evaluated the pmTOR and pEIF4EBP1 (the downstream effector protein of mTORC).The data implied SH2 TrM-(Arg)9 had no noticeable effect on the phosphorylation and total levels of mTOR or EIF4EBP1 in PANC-1 cells (Fig. 2J).These findings indicated SH2 TrM-(Arg)9-mediated autophagy inhibition might be independent of the mTOR signaling pathway, but a result of other signaling cascades.

SH2 TrM-(Arg)9 mediated autophagy inhibition of PDAC cells through affecting phosphotyrosines
The aberrant activation of tyrosine phosphorylation has been reported to play an essential role in human specimens and PDAC cells [18].To unveil the molecular mechanism underlying the physiological activities, we systematically investigated the global phosphorylation events of PANC-1 cells caused by SH2 TrM-(Arg)9 through label-free quantitative phosphoproteomics [33], expecting to figure out the critically altered tyrosine phosphorylation of  proteins during the anti-tumor process.After three independent experiments, we found that Y792 of DDR1 (a member of the RTK family) showed a consistent trend of change in the three sources of PANC-1 cells, which indicated the importance of this site (Dataset 1).DDR1 plays a role in neurological disorders during the autophagy process [34,35].In pancreatic cancer, blocking the collagen/DDR1-based signaling pathway inhibits tumor growth [36,37]; however, its effect on autophagy remains unknown.From phosphoproteomics results, we found the level of phosphotyrosines of DDR1(Y792) was significantly decreased in SH2 TrM-(Arg) 9 treated groups (Fig. 3A, B).To further confirm the significance of the phosphotyrosines-related signaling pathways, we measured the phosphorylation levels of tyrosine (pY) and DDR1.The data indicated SH2 TrM-(Arg)9 downregulated the phosphorylation levels of tyrosine residues (Fig. 3C, D).We further measured the phosphorylation levels of total tyrosine and DDR1(Y792) in PDAC and adjacent tissues from patients.Using immunostaining, we found both pY and pDDR1(Y792) levels elevated in tumor tissues (Fig. 3E, F).
What is more, we also found that JAK/STAT signaling pathway had little effect on PDAC cells autophagy in our study.Altered JAK/ STAT expression has been found to be accompanied by the abnormal autophagy activity in many oncological studies [43,44].In Fig. 1L, we have found SH2 TrM-(Arg)9 could block the JAK/ STAT signaling pathway, but it is interesting that SH2 TrM-(Arg)9 not Tofacitinib (JAK/STAT inhibitor) restrain autophagy of PDAC cells (Fig. S1I).So, we considered other signaling pathway but not JAK/STAT engaged in the regulation of autophagy.All these results above inspired us that DDR1-mediated signaling pathway may regulate autophagy of PDAC cells.

Combination of SH2 TrM-(Arg)9 and GEM exhibits satisfactory anti-tumor efficacy in vivo
To further confirm that the inhibition of autophagy is a potential therapeutic approach against PDAC in vivo, PANC-1 and PANC-1/  GEM cells were inoculated subcutaneously into NOD/SCID mice.Compared with the tumor volume of mice inoculated with the PBS and GEM control groups, (SH2 TrM-(Arg)9 + GEM) markedly reduced the tumor volumes (Fig. 6A-D).The histological data showed (SH2 TrM-(Arg)9 + GEM) resulted in a higher level of necrotic lesions, lower level of proliferation ability, and higher apoptosis rate than the other groups (Fig. 6E, F).This result indicated SH2 TrM-(Arg)9 enhanced the anti-tumor effect of GEM.Moreover, p62 and LC3-II/LC3-I ratio increased in tumors in SH2 TrM-(Arg)9 and (SH2 TrM-(Arg)9 + GEM)-treated groups (Fig. 6G,  H). mTOR signaling was also not influenced simultaneously and was consistent with the data in cells (Fig. 6I), meaning that SH2 TrM-(Arg)9 affected autophagy without conditional regulation of the mTOR signaling pathway.Treatment with (SH2 TrM-(Arg) 9 + GEM) decreased pDDR1, pPYK2, and pERK1/2 of tumors in PANC-1 and PANC-1/GEM mouse models (Fig. 6I).Therefore, SH2 TrM-(Arg)9 boosted the susceptibility of PDAC cells to GEM, thereby enhancing the efficacy of GEM against pancreatic cancer.

DISCUSSION
Autophagy is a widespread self-digestion process that has been retained in the evolution of organisms, through which cells can meet the needs of metabolism and organelle renewal [49].In the environment of hypoxia, nutritional deficiency, metabolic stress, and anticancer treatment, cancer cells will survive by autophagy [50].Our study confirmed activation of collagen-DDR1-mediated signaling promoted autophagic flux in pancreatic cancers and prolonged the survival of PDAC cells.We also identified DDR1, together with the 14-3-3/Beclin1/Akt1 protein complex, is required for collagen-mediated DDR1/PYK2/ERK signaling, whereas SH2 TrM-(Arg)9 inhibited autophagic flux and regulated autophagyassociated therapy sensitivity (Fig. 7).Our experiments uncovered a DDR1-driven mechanism that eliminates PDAC therapy resistance, and laid the groundwork for the development of therapysensitizing agents.
Compared with the SH2 domain, the SH2 superbinder has a stronger binding ability for the pY site and further regulates the activation process of the cell [17].We pioneered the integration of SH2 superbinder with a cell penetrating peptide (CPP), to allow it to pass through cell membranes and SH2 TrM-(Arg)9 has a wide range of anticancer effects in melanoma, pancreatic cancer, nonsmall cell lung cancer, and other cells [17,18,51,52].The specific mechanism of action involves cell proliferation, migration, and apoptosis [17,18].What's more, SH2 TrM-(Arg)9 has little toxic effect on normal cells.It is a promising broad-spectrum cancer drug.Our group has long maintained the improvement of the function and safety of SH2 superbinder [17,18,51,52].Here we focused on the relationship between SH2 superbinder and autophagy.Other researchers have achieved treatment of pancreatic cancer by inhibiting autophagy [13,27].In light of these previous findings, we hypothesized and was able to verify SH2 superbinder affected autophagy by regulating the DDR1/ PYK2/ERK signaling.Since the SH2 superbinder used in this study did not conjugate specific aptamers [18] targeting tumor cells, its targeting ability could only rely on the EPR effect (enhanced permeability and retention effect) of the tumor.
Additionally, SH2 TrM-(Arg)9 significantly reduced the side effects of GEM and increased the sensitivity of cells to GEM in vitro and in vivo.To ensure the synergistic anti-tumor effect of GEM, a lower dose was used to reduce its toxicity and side effects (Fig. S5).Studies on conjugating aptamers have been described in our previous paper [18].This study focuses on revealing the effect of the SH2 superbinder on autophagy, so there are no experiments on targeting.
Post-translational modifications of proteins, such as phosphorylation, acetylation, and ubiquitination, are often associated with the autophagy process, and phosphorylation mainly focuses on serine and threonine [53].Although tyrosine phosphorylation accounts for a small proportion of protein phosphorylation, it also plays an important role in cancer development [54].However, the relationship between autophagy and phosphorylation has been less studied [55].
In conclusion, we analyzed the mechanism of action of the SH2 superbinder in-depth.For the first time, SH2 superbinder was found to have an anticancer role by affecting autophagy in PDAC cells and animal models.When combined with GEM, SH2 superbinder reduced chemotherapy resistance and side effects.Based on the significant autophagy inhibition effect of SH2 superbinder on PDAC cells, we provided evidence to support using SH2 superbinder as an anticancer drug once again, providing a new direction for the clinical treatment of PDAC.
All amplified products were purified using the Gel Band Purification Kit (Beijing ComWin Biotech).The backbone was digested with BamHI (Cat.#R3136V) and HindIII (Cat.#R3104V) at 37 °C for 2 h, and the purified PCR products were reacted with the linearized pGEX-4T3 vector at 37 °C for 30 min.The experiment used the One Step Cloning Kit (Vazyme, Nanjing), according to the principle of homologous recombination.
The specific procedure of plasmid transformation, protein expression and purification can refer to the previous study [17].The BCA Protein Assay Kit was used to quantify the protein concentration in cell homogenates.Immediately prior to use in biological assays, protein purity was verified by SDS-PAGE using Coomassie brilliant blue staining intensity.

Transmission electron microscopy
Cells from different groups were fixed with 2.5% glutaraldehyde solution according to the previous description [56].Images were captured by a transmission electron microscope (TEM, JEOL, Tokyo, Japan).

In vitro DDR1 kinase assay
The specific DDR1 kinase activity in the presence and absence of SH2 TrM-(Arg)9 was measured using a Universal Kinase Activity Kit (R&D Systems, Cat.#EA004).The assay measures the inorganic phosphate that is released from ADP via substrate phosphorylation by DDR1.The specific procedures are carried out according to the instructions.

Patients and sample collection
PDAC and the adjacent tissues were obtained from patients who underwent surgical resection for PDAC at the Tongji Hospital (Wuhan, China).Tissue acquisition and handling of human tissue specimens used in this study have been approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology.Patient information on clinical specimens is listed in Table S3.

Statistics
Results are presented as mean ± standard deviation (SD) and analyzed.The student's t test was used for statistical significance of the differences between the different groups.P values of less than 0.05 were considered statistically significant.*P < 0.05, **P < 0.01 and ***P < 0.001.
Other methods are provided in Supplementary File 1.